Labelled Silica Nanoparticles for Immunochromatographic Reagent, Immunochromatographic Test Strip Using the Same, and Immunochromomatographic Fluorescence-Detecting System or Radiation-Detecting System

ABSTRACT

Labelled silica nanoparticles for immunochromatographic reagent, comprising silica nanoparticles containing a labelled substance.

CROSS REFERENCE

This application is a Divisional of co-pending application Ser. No.13/095,196 filed on Apr. 27, 2011, which is a Divisional of Ser. No.12/155,622 filed Jun. 6, 2008 (now U.S. Pat. No. 7,955,866), the entirecontents of which are hereby incorporated by reference, and to whichpriority is claimed under 35 U.S.C. §120. Priority is also claimed,under 35 U.S.C. §119, to Japanese Application No. 2007-153366 filed Jun.8, 2007, the entire contents of which are hereby incorporated byreference.

FIELD

The present invention relates to silica nanoparticles containing alabelled substance and an immunochromatographic reagent allowingsimultaneous multi-item detection by using the same. The presentinvention also relates to an immunochromatographic test strip using theimmunochromatographic reagent, and an immunochromatographicfluorescence-detecting system employing a fluorescent substance or aradioactive substance as the labelled substance or a radiation-detectingsystem employing a radioactive substance as the labelled substance thatallows high-sensitivity detection.

BACKGROUND

The immunochromatographic method is an immunological test method ofdetermining the presence or absence of an analyte substance by makingthe analyte substance captured by labelled particles move through aporous support in capillary phenomenon, efficiently bringing thelabelled particles into contact with a capturing substance locally(e.g., linearly) immobilized on the porous support and thus,concentrating the analyte substance, and making the immobilized linecolored by the capturing substance. The immunochromatographic method hasthe following three characteristics:

(1) Rapid test is possible, as the period needed for test is 20 minutesor less.

(2) Multi-item analysis is possible, because the operation was simpleand easy, demanding only dropwise application of the sample.

(3) No special detector is needed and the test is simple and easy,allowing test by general users.

Because of these characteristics, the immunochromatographic method hasbeen applied as a pregnancy test reagent and an influenza test reagent,and attracting attention as a new POCT (Point Of Care Testing) method.

The POCT is a test for diagnosis that is carried out at a place as closeto the patient as possible. Conventionally, samples collected such asblood, urine and infected organs were sent to the central laboratory ofhospital or a professional laboratory for analysis for data, and thus, along period (e.g., at least one day) was needed before final diagnosis.The POCT above promises rapid and accurate therapy, based on testinformation obtained instantaneously and allows emergency tests inhospital and during operation, and thus, there is an increased need forit recently in the medical settings.

Currently, gold nanoparticles are used most frequently as the labelledsubstance in the immunochromatographic method. The gold nanoparticleshave a large absorbance and are thus superior in visibility, but haveonly a single color, consequently causing a problem of restriction ofthe color for use. In addition, gold particles having a larger particlediameter often aggregate and blacken over time, causing a problem thatit was difficult to make the gold particles develop a clear color.

SUMMARY

The present invention resides in labelled silica nanoparticles for animmunochromatographic reagent, comprising silica nanoparticlescontaining a labelled substance.

Further, the present invention resides in an immunochromatographicreagent, comprising the labelled silica nanoparticles.

Further, the present invention resides in an immunochromatographic teststrip, comprising a sample application member, a member impregnated withthe labelled silica nanoparticles as the immunochromatographic reagent,a membrane having an antibody-immobilized region, and an absorption padthat are connected in series.

Further, the present invention resides in an immunochromatographicfluorescence-detecting system, comprising the immunochromatographicreagent, wherein the wavelength of an excitation light source used fordetection of the fluorescence emitted from the silica nanoparticles is200 nm to 600 nm.

Further, the present invention resides in an immunochromatographicradiation-detecting system, comprising the immunochromatographicreagent, wherein radiation emitted from the silica nanoparticles ismeasured with a Geiger counter or by photosensitizing an X-ray film.

Other and further features and advantages of the invention will appearmore fully from the following description, with taken in connection withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a top view illustrating an immunochromatographic test stripof the present invention, and FIG. 1 b is a cross-sectional view of theimmunochromatographic test strip in the top view shown in FIG. 1 a.

FIG. 2 is a TEM micrograph showing the labelled substance-containingsilica nanoparticles obtained.

DETAILED DESCRIPTION

According to the present invention, there is provided the followingmeans.

(1) Labelled silica nanoparticles for an immunochromatographic reagent,comprising silica nanoparticles containing a labelled substance.

(2) The labelled silica nanoparticles for an immunochromatographicreagent according to the item (1), wherein the labelled substance is afluorescent substance or a light-absorbing substance.

(3) The labelled silica nanoparticles for an immunochromatographicreagent according to the item (1), wherein the labelled substance is aradioactive substance.

(4) The labelled silica nanoparticles for an immunochromatographicreagent according to any one of the items (1) to (3), wherein theaverage diameter of the labelled silica nanoparticles is 20 to 1000 nm.

(5) The labelled silica nanoparticles for an immunochromatographicreagent according to any one of the items (1) to (4), wherein thenanoparticles contain no surfactant.

(6) The labelled silica nanoparticles for an immunochromatographicreagent according to any one of the items (1) to (5), wherein thenanoparticles are surface-modified with an analyte-recognizingsubstance.

(7) The labelled silica nanoparticles for an immunochromatographicreagent according to the item (6), wherein the analyte-recognizingsubstance is an antibody.

(8) The labelled silica nanoparticles for an immunochromatographicreagent according to the item (7), wherein the antibody binding amountto a surface area of 1 m² of the silica nanoparticles is 0.35 mg to 7mg.

(9) An immunochromatographic reagent, comprising the labelled silicananoparticles according to any one of the items (6) to (8).

(10) The immunochromatographic reagent according to the item (9),wherein two or more kinds of analytes are detected simultaneously byusing two or more kinds of labelled silica nanoparticles distinguishedfrom each other by difference in the kind and the content of thelabelled substance.

(11) The immunochromatographic reagent according to the item (9) or(10), wherein the labelled substance is a light-absorbing substance thathas a maximum absorption wavelength in the absorption spectrum thereofin the range of 200 to 800 nm.

(12) The immunochromatographic reagent according to the item (11),wherein the molar extinction coefficient of the silica nanoparticles atthe maximum wavelength in the absorption spectrum is 5×10⁷M⁻¹ cm⁻¹ ormore.

(13) The immunochromatographic reagent according to the item (9) or(10), wherein the labelled substance is a fluorescent substance and thereagent allows high-sensitivity detection by detection of fluorescenceemitted from the silica nanoparticles containing the fluorescentsubstance.

(14) The immunochromatographic reagent according to the item (13),wherein the fluorescence is blue fluorescence, yellow fluorescence,orange fluorescence or red fluorescence.

(15) An immunochromatographic test strip, comprising a sampleapplication member, a member impregnated with the labelled silicananoparticles as the immunochromatographic reagent according to any oneof the items (9) to (14), a membrane having an antibody-immobilizedregion, and an absorption pad that are connected in series.

(16) The immunochromatographic test strip according to the item (15),wherein two or more kinds of analytes are detected simultaneously, basedon the difference in hue of color formation or fluorescence wavelengthin a single antibody-immobilized region.

(17) An immunochromatographic fluorescence-detecting system, comprisingthe immunochromatographic reagent according to the item (13) or (14),wherein the wavelength of an excitation light source used for detectionof the fluorescence emitted from the silica nanoparticles is 200 nm to600 nm.

(18) The immunochromatographic fluorescence-detecting system accordingto the item (17), further comprising a filter transmitting theexcitation light at a particular wavelength from the excitation lightsource and a filter eliminating the excitation light and transmittingonly the fluorescence.

(19) The immunochromatographic fluorescence-detecting system accordingto the item (18), further comprising a photoelectron multiplier or a CCDdetector detecting the fluorescence, wherein the system allowsquantitative determination of the analyte by detection of thefluorescence and measurement of the fluorescent intensity with thephotoelectron multiplier or the CCD detector.

(20) The immunochromatographic fluorescence-detecting system accordingto any one of the items (17) to (19), wherein the excitation lightsource is a mercury lamp, a halogen lamp, or a xenon lamp.

(21) The immunochromatographic radiation-detecting system, comprisingthe immunochromatographic reagent according to the item (9), whereinradiation emitted from the silica nanoparticles is measured with aGeiger counter or by photosensitizing an X-ray film.

First, the labelled silica nanoparticles for an immunochromatographicreagent of the present invention will be described.

The labelled silica nanoparticles for an immunochromatographic reagentof the present invention comprise silica nanoparticles containing alabelled substance.

In the present invention, the “silica nanoparticles” mean colloidalsilica particles having an average diameter of 1,000 nm or less.

Generally, silica refers to a three-dimensional structure consisting ofsilicon and oxygen atoms forming siloxane bonds (Si—O bonds) with eachother, but in the present invention, it also includes three-dimensionalstructures of silicon and oxygen atoms containing organosiloxanecomponents.

In the present invention, the average diameter of the labelled silicananoparticles is preferably 20 to 1000 nm, more preferably 20 to 600 nm,furthermore preferably 60 to 300 nm. An excessively smaller particlediameter leads to deterioration in detection sensitivity, while anexcessively large particle diameter may cause clogging of the poroussupport (membrane) used in the immunochromatographic method.

In the present invention, the average diameter is an average diameter ofthe circle (average circle-equivalent diameter) obtained by measuringthe total projected area of 50 randomly-selected silica nanoparticles inan image obtained under transmission electron microscope (TEM), scanningelectron microscope (SEM) or the like using an image processingequipment, dividing the total area with the number of the silicaparticles (50), and determining the circle having an area equivalent tothat.

The variation coefficient, so-called CV value, of the particle sizedistribution is not particularly limited, but preferably 10% or less,more preferably 8% or less.

In the present description and the claims thereof, a term“monodispersion” is used for particles having a CV value of 15% or less.

In the present invention, the silica particle to be used is notparticularly limited, and silica particles produced by any preparationmethod may be used. Examples thereof include the silica particlesprepared by the sol-gel process, for example as described in Journal ofColloid and Interface Science, 159, 150-157 (1993).

It is particularly preferred that silica nanoparticles containing thelabelled substance prepared according to a method of preparing colloidalsilica particles containing a fluorescent dye compound described inWO2007/074722A1 are used.

Specifically, the silica nanoparticles containing the labelled substancecan be prepared by:

(a) obtaining a product by reacting the labelled substance with asilane-coupling agent to form a chemical bond, such as a covalent bond,an ionic bond or the like, or to adsorb the labelled substance onto thesilane-coupling agent, and

(b) polymerizing one or more silane compounds to the product.

As the preferred embodiment of the method of preparing silicananoparticles containing the labelled substance, the silicananoparticles containing the labelled substance can be prepared by:

(a) obtaining a product by allowing the labelled substance having anactive group such as N-hydroxysuccinimide (NHS) ester group, a maleimidegroup, an isocyanate group, an isothiocyanate group, an aldehyde group,a para-nitrophenyl group, a diethoxymethyl group, an epoxy group, or acyano group, to react with a silane-coupling agent having a substituentgroup (such as amino group, hydroxyl group, or thiol group) reactivewith the active group, to form a covalent bond, and

(b) polymerizing one or more silane compounds to the product.

Specific examples of the active group-containing the labelled substancesinclude NHS ester group-containing labelled substances such as 5- (and6)-carboxytetramethylrhodamine succinimidyl ester (trade name,manufactured by emp Biotech GmbH).

Specific examples of the radioactive substances having such an activegroup include NHS ester derivatives of carboxyl group-containingradioactive substances, such as Phenylacetic acid, [acetate-1,2-14C](product name, manufactured by Daiichi Pure Chemicals Co. Ltd.),d-[8,9-3H] Biotin (product name, manufactured by GE HealthcareBioscience Co. Ltd.), d-[carbonyl-14C] Biotin (product name,manufactured by GE Healthcare Bioscience Co. Ltd.), [1-14C] stearic acid(product name, manufactured by GE Healthcare Bioscience Co. Ltd.) andthe like.

Examples of the silane-coupling agent having a substituent group includean amino group-containing silane-coupling agent, such asγ-aminopropyltriethoxysilane (APS),3-[2-(2-aminoethylamino)ethylamino]propyltriethoxysilane,N-2-(aminoethyl)-3-aminopropylmethyldimethoxysilane or3-aminopropyltrimethoxysilane. Among them, APS is preferable.

The silane compound to be polymerized is not particularly limited, andexamples thereof include tetraethoxysilane (TEOS),γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane,γ-aminopropyltriethoxysilane (APS), 3-thiocyanatopropyltriethoxysilane,3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane,and 3-[2-(2-aminoethylamino)ethylamino]propyltriethoxysilane. Amongthem, TEOS, MPS, and APS are preferable.

A group of spherical or almost spherical silica particles can beprepared by the method described above. Specifically, the almostspherical fine particle means a particle having a major axis/minor axisratio of 2 or less.

For obtaining silica particles having a desired average diameter, it ispreferable to perform filtration by using an ultrafiltration membranesuch as YM-10 or YM-100 (trade names, manufactured by Millipore) forremoval of particles having excessively larger and smaller particlediameters or to perform centrifugation at a suitable gravitationalacceleration for recovery only of the supernatant or sediment.

Conventional nanoparticles such as latex particles are lower indispersion stability, and copresence of a surfactant was needed fordispersion thereof in an aqueous solution such as buffer solution (see,for example, Masasumi Koishi and Takao Iwasaki, “Preparation of polymerparticles,” Ed., by Soc. Polymer Science, Japan, published by KyoritsuShuppan, 1994). When conventional nanoparticles such as latex particlesare used as a labelling reagent such as the immunochromatographicreagent, it was known that the surfactant exerted an adverse effect asan impurity on the S/N ratio (Signal/Noise ratio).

On the other hand, the immunochromatographic labelled silicananoparticles of the present invention is superior in dispersionstability in aqueous solutions such as buffer solution and can bedispersed without use of a surfactant. Therefore, there is no need tocontain the surfactant for the dispersion. Thus, there is no adverseeffect by the surfactant generated when the nanoparticles are used as animmunochromatographic reagent.

In the present invention, the labelled substance is immobilized in thelabelled silica nanoparticles. The labelled substance is, for example, afunctional substance such as a fluorescent substance, a light-absorbingsubstance, a radioactive substance. The fluorescent substance, thelight-absorbing substance or the radioactive substance is notparticularly limited, and, for example, an organic molecule, aninorganic compound, a semiconductor particle, or the like. When thelabelled substance is a fluorescent substance, a light-absorbingsubstance or a radioactive substance, the concentration of thefluorescent substance, the light-absorbing substance or the radioactivesubstance in the labelled silica nanoparticles is preferably 20 mmol/lor more, more preferably 40 to 80 mmol/l.

Herein, the “concentration of the fluorescent substance, thelight-absorbing substance or the radioactive substance in the labelledsilica nanoparticles” is a value obtained by dividing the molar numberof the fluorescent substance, the light-absorbing substance or theradioactive substance by the volume of the labelled silicananoparticles. The molar number of the fluorescent substance or thelight-absorbing substance is determined from the absorbance orfluorescence intensity of the labelled silica nanoparticles.

Further, the molar number of the radioactive substance was determinedfrom the radiation quantity from the labelled silica nanoparticles.

Further, the volume of the labelled silica nanoparticles is obtained byseparating the labelled silica nanoparticles from the labelled silicananoparticles dispersion by centrifugation or ultrafiltration, dryingand weighing the particles, and dividing the mass by the silica particledensity of 2.3 g/cm³.

For use of the labelled silica nanoparticles in immunochromatographicmethods, the silica nanoparticles are preferably surface-modified withan analyte-recognizing substance (e.g., biological molecule such asantibody, antigen, DNA, or RNA) and the silica nanoparticles are morepreferably surface-modified with antibody.

When the analyte-recognizing substance (the substance recognizing theanalyte) is an antibody, the antibody binding amount on a surface areaof 1 m² of the silica nanoparticle is preferably 0.35 mg to 7 mg, morepreferably 0.7 mg to 3.5 mg.

The binding amount of the analyte-recognizing substance to the silicananoparticles is determined by measuring the analyte-recognizingsubstance contained in the solution, which is obtained by removal ofparticles from the surface modification reaction solution bycentrifugation or ultrafiltration, quantitatively by a common proteinanalysis method (e.g., BCA (bicinchoninic acid) method, UV method, Lowrymethod, or Bradford method) and also measuring the amount of theanalyte-recognizing substance decreased.

In the present specification and claims, the analyte-containing samplesolution is not particularly limited, and examples thereof includeurine, blood and the like.

In the present specification and claims, the analytes to be detected,quantified, tested, diagnosed or judged include antigens, antibodies,DNAs, RNAs, saccharides, sugar chains, ligands, receptors, peptides,chemicals and the like. More specifically, an important pregnancy markerantigen, human gonadotropin (hCG)-peptide hormone in urine, is tested asan analyte for evaluation or diagnosis of pregnancy.

The method of binding the analyte-recognizing substance to the labelledsilica nanoparticles is not particularly limited, and thus, theanalyte-recognizing substance may be adsorbed on the labelled silicananoparticles, for example, by electrostatic attractive force, Van derWaals force, hydrophobic interaction, or the like, or bonded theretochemically by using a crosslinking or condensation agent. Yetalternatively, a thiol group may be introduced onto the surface of thelabelled silica nanoparticles by using a thiol group-containingsilane-coupling agent such as MPS (γ-mercaptopropyltriethoxysilane) andbound to the thiol group of the analyte-recognizing substance by forminga S—S bond.

If the particles aggregate when an analyte-recognizing substance, suchas the biological molecule (e.g., antibody, antigen, DNA, or RNA) on thelabelled silica nanoparticle surface described above, is bound thereto,the labelled silica nanoparticle surface may be previouslysurface-treated by alternating adsorption method. The alternatingadsorption method is a method of forming a polymer thin film on thesurface of a charged substrate or particles by allowing adsorption of acharged polymer thereon by electrostatic attractive force. It ispossible to charge the particle surface by alternating adsorption on thesurface of the labelled silica nanoparticles, which generateselectrostatic repulsive force between particles and improvesdispersibility. In addition, the polymer bound to the particles, whichhas an excluded volume, also improves dispersion by steric repulsionforce.

Hereinafter, the immunochromatographic reagent of the present inventionwill be described.

The immunochromatographic reagent of the present invention comprises thelabelled silica nanoparticles, and is preferably surface-modified withan analyte-recognizing substance, as described above, and is morepreferably surface-modified with antibody.

The immunochromatographic reagent of the present invention is preferablysilica nanoparticles containing a light-absorbing substance, afluorescent substance or a radioactive substance as the labelledsubstance as described above.

The light-absorbing substance is not particularly limited, if it is asubstance absorbing light from any light source described above, but,for detection with a commonly-used detector such as plate reader (e.g.,Vmax (trade name: manufactured by Molecular Devices), micro plate readerMPR-A4i (trade name: manufactured by Toso Corporation)) and from theviewpoint of data compatibility, a light-absorbing substance having amaximum absorption wavelength of absorption spectrum in the range of 200to 800 nm is preferable, and a light-absorbing substance having amaximum absorption wavelength of absorption spectrum in the range of 400to 700 nm is more preferable.

The “maximum absorption wavelength” herein is the wavelength of the peakhaving the strongest absorption among the absorption peaks when thereare multiple absorption peaks present in the absorption spectrum.

Any light-absorbing dye may be used, if it has a maximum absorptionwavelength of absorption spectrum in the range of 200 to 800 nm, but,for example, an NHS ester of DYQ-660 (trade name, manufactured byDyomics GmbH) represented by the following Formula is preferable.

The silica nanoparticles containing the light-absorbing substance has amolar extinction coefficient ε of 5×10⁷ M⁻¹ cm⁻¹ or more, and ε ispreferably 2×10⁸ M⁻¹ cm⁻¹ to 1×10¹¹ M⁻¹ cm⁻¹.

The absorbance, absorption spectrum and ε of the silica nanoparticlescontaining a light-absorbing substance may be determined by using anyabsorptiometer or plate reader, as the silica nanoparticles aredispersed, for example, as aqueous dispersion, ethanol dispersion, orN,N-dimethylformamide dispersion.

The “molar extinction coefficient ε of the silica nanoparticlescontaining a light-absorbing substance” is a molar extinctioncoefficient ε of the silica nanoparticles containing a light-absorbingsubstance in dispersion, as determined by measuring the absorbance ofthe dispersion of the silica nanoparticles containing a light-absorbingsubstance and calculating according to the Lambert-Beer's equation.

In the immunochromatographic reagent of the present invention, when usedare silica nanoparticles obtained by surface-modification of the silicananoparticles containing a fluorescent substance as the labelledsubstance with an analyte-recognizing substance, it is possible toachieve high-sensitivity detection or quantitative determination, bydetecting the fluorescence emitted from the silica nanoparticles.

The fluorescent substance is not particularly limited, but for detectionwith a commonly-used detector (e.g., AE-6931FXCF Printgraph (trade name,manufactured by ATTO)) and from the viewpoint of data compatibility, afluorescent substance emitting blue fluorescence (440 to 490 nm), yellowfluorescence (540 to 590 nm), orange fluorescence (590 to 620 nm) or redfluorescence (620 to 740 nm) is preferable.

Hereinafter, the immunochromatographic test strip of the presentinvention will be described.

The immunochromatographic test strip of the present invention has

(1) a sample application member (sample pad) and a member impregnatedwith the labelled silica particles (conjugate pad), and

(2) the conjugate pad and a membrane having an antibody-immobilizedregion (antibody-immobilized membrane), and

(3) that are connected to each other in series, so that there iscapillary phenomenon generated between the antibody-immobilized membraneand the absorption pad.

A preferable embodiment of the immunochromatographic test strip of thepresent invention will be described with reference to FIGS. 1 a and 1 b.

FIG. 1 a is a top view illustrating the immunochromatographic test stripof the present invention, and FIG. 1 b is a cross-sectional view of theimmunochromatographic test strip in the top view.

The immunochromatographic test strip 1 of the present inventionpreferably has a sample application member (sample pad) 2, a labelledsilica nanoparticle-impregnated member (conjugate pad) 3, anantibody-immobilized membrane 4, and an absorption pad 5. Eachconstituent member is more preferably supported by a backing sheet 6with adhesive.

The antibody-immobilized region of the membrane 4 preferably has a testline 41 where a capturing antibody for evaluation of presence or absenceof an analyte, i.e., for judgment of positivity or negativity, isimmobilized and a control line 42 where antibodies for capturing allanalytes labelled by the labelled silica particles are immobilized.

Hereinafter, each of the members above will be described.

1) Sample Application Member (Sample Pad) 2

The sample pad 2 is a constituent member where an analyte-containingsample is applied dropwise.

2) Labelled Silica Nanoparticle-Impregnated Member (Conjugate Pad) 3

The conjugate pad 3 is a constituent member impregnated with thelabelled silica nanoparticles, where the analyte contained in the samplesolution moving from the sample pad 2 by capillary phenomenon iscaptured and labelled by the labelled silica particles by molecularrecognition reaction such as antigen-antibody reaction.

The content of the labelled silica nanoparticles per unit area (cm²) ofthe conjugate pad 3 is not particularly limited, but preferably 50 μg to2 mg. The impregnation may be effected, for example, by coating,dropwise application, or spraying of the dispersion of the labelledsilica particles and subsequent drying.

3) Antibody-Immobilized Membrane 4

The membrane 4 is a constituent member where the analyte labelled by thesilica nanoparticles moves by capillary phenomenon and which has anantibody-immobilized region (judgment region) carrying out a reactionforming a sandwiched immunocomplex in the form of immobilizedantibody-analyte-labelled silica nanoparticle.

The shape of the antibody-immobilized region (judgment region) in themembrane is not particularly limited, if the capturing antibody isimmobilized locally there, and may be linear, circular, belt-shaped andthe like in shape, but preferably linear, more preferably linear with awidth of 0.5 to 1.5 mm.

The analyte labelled with silica particles can be captured in theantibody-immobilized region (judgment region) by the reaction forming ansandwiched immunocomplex in the form of immobilizedantibody-analyte-labelled silica nanoparticles, and it is possible todetermine presence or absence of the analyte, i.e., to determinepositivity or negativity, by the degree of the coloring or fluorescencederived from the silica nanoparticles on the complex formed. Thus, thelabelled silica particles are concentrated in the antibody-immobilizedregion (judgment region), allowing detection and judgment visually ascoloring signal or by analysis with a detecting device.

For sufficient completion of the sandwiched immunocomplex-formingreaction or for prevention of the influence on measurement by thecoloring substance in the liquid sample and by the labelled silicananoparticles not bound to the analyte, the judgment region in themembrane is preferably formed as it is separated from the end connectedto the conjugate pad and also from the end connected to the absorptionpad to some extent (e.g., located in the middle of the membrane).

The amount of the antibody immobilized in the antibody-immobilizedregion (judgment region) is not particularly limited, but when the shapeof the antibody-immobilized region is linear, it is preferably 0.5 μg to5 μg per unit length (cm). The immobilization may be performed, forexample, a method of physical adsorption by coating, dropwiseapplication or spraying of the antibody solution and subsequent drying.

For prevention of the influence on measurement by nonspecific adsorptionafter the antibody immobilization described above, the membrane ispreferably subjected to so-called blocking treatment entirely. Forexample, the membrane is immersed in a buffer solution containing ablocking agent such as albumin, casein or polyvinylalcohol for asuitable period and then dried. Examples of the commercially availableblocking agents include Skimmilk (manufactured by DIFCO), 4% Block Ace(manufactured by Meiji Dairies Corp.) and the like.

4) Absorption Pad 5

The absorption pad 5 is a constituent member generating a particularflow by absorbing the sample solution and the labelled silica particlesmoving through the membrane by capillary phenomenon.

The material for each constituent member is not particularly limited,and any material used for immunochromatographic test strip may be used,but a glass fiber pad such as Glass Fiber Conjugate Pad (trade name:manufactured by MILLIPORE) is preferable as the sample pad and theconjugate pad; a nitrocellulose membrane such as Hi-Flow Plus120membrane (trade name: manufactured by MILLIPORE) is preferable as themembrane; and a cellulose membrane such as Cellulose Fiber Sample Pad(trade name: manufactured by MILLIPORE) is preferable as the absorptionpad.

The backing sheet with adhesive is, for examples, AR9020 (trade name,manufactured by Adhesives Research).

The test strip may be prepared by placing a sample application member(sample pad), a labelled silica particle-impregnated member (conjugatepad), an antibody-immobilized membrane, and an absorption pad in thatorder (preferably on a backing sheet), while bonding one end of eachunit onto the end of the next member with an overlapping a width ofapproximately 1 to 5 mm to cause capillary phenomenon between respectivemembers.

Needless to say, the degree of color development can be determined byvisual observation and also by using a tester for urine test paper suchas Pretester RM-405 or Pretester RM-505 (trade names, manufactured byWako Pure Chemical Industries, Ltd.), a densitometer, or the like.

The test strip is preferably placed in a housing (casing), for example,of a plastic material with an observation window for visual examinationof the detection line of test strip, for easier operation by generalusers who are not skilled in operation and from the point of POCT. Forexample, the housing described in JP-A-2000-356638 (“JP-A” meansunexamined published Japanese patent application) and the like are usedfavorably.

The labelled silica nanoparticles as the immunochromatographic reagentof the present invention can emit lights in various hues (colors) and atvarious fluorescence wavelengths, by containing different kinds oflabelled substances. By using two or more of the labelled silicananoparticles thus obtained it is possible, for example, to detect,quantify, judge or diagnose two or more analytes simultaneously in oneoperation on the test strip.

Then, the two or more labelled silica nanoparticles should besurface-modified respectively with substances recognizing differentanalytes molecularly.

Specific examples thereof include an immunochromatographic reagentcomprising red dye (e.g., rhodamine)-containing silica nanoparticlessurface-modified with a mouse anti-influenza A virus nucleoproteinmonoclonal antibody that recognizes influenza A virus nucleoprotein andblue dye (e.g., DYQ-660 (trade name, manufactured byDyomics))-containing silica nanoparticles surface-modified with a mouseanti-influenza B virus nucleoprotein monoclonal antibody that recognizesinfluenza B virus nucleoprotein, and a test strip by using the same.

With the immunochromatographic reagent containing two or more labelledsilica nanoparticles described above and the test strip by using thesame, it is possible to detect, quantify, judge or diagnose two or moreanalytes simultaneously based on the difference in hue of colorformation or in fluorescence wavelength in the same antibody-immobilizedregion. The same antibody-immobilized region is preferably linear inshape.

The color (hue) and the emission wavelength of conventionalnanoparticles (e.g., gold nanoparticle), which often emits asingle-colored light or a fluorescence at a single wavelength, were veryrestricted. Therefore, when such conventional nanoparticles are used inthe immunochromatographic method, it was substantially difficult todetect or judge two or more analytes, and thus, needed to formantibody-immobilized regions (judgment regions) carrying differentimmobilized antibodies on a membrane at different positions. Whenmultiple antibody-immobilized regions are formed at positions close toeach other, the color formation or emission signal from analytes oftencause confusion, because the regions emit a light in a single colors ora fluorescent light at a single wavelength.

In contrast, the labelled silica nanoparticles can be provided bycontaining different kinds of labelled substance with various hues,various fluorescence wavelengths and the like, and thus, it is possibleto detect, quantify, judge or diagnose multiple kinds of analytessimultaneously, as multiple kinds of antibodies are immobilized in asingle antibody-immobilized region in the test strip of the presentinvention.

Specifically, for example, when analytes A and B are to be measured, animmobilized test strip having an a single antibody-immobilized regioncarrying immobilized antibodies to the analytes A and B is prepared byusing blue dye (e.g., DYQ-660 (trade name, manufactured byDyomics))-containing silica nanoparticles surface-modified with ananalyte A-recognizing antibody or the fragment thereof and yellow dye(e.g., fluorescein)-containing silica nanoparticles surface-modifiedwith an analyte B-recognizing antibody or the fragment thereof. It ispossible to decide whether the analytes are present, by applying anunknown sample solution possibly containing the analytes A and Bdropwise on the sample pad of the test strip and observing theantibody-immobilized region. In the specific example above, blue hue(color) of the reaction line indicates major presence of the substanceA, while yellow color, of the substance B, and when the analytes A and Bare present in almost the same amounts, a reaction line green in coloris observed. In this way, it is possible to determine the abundanceratio of the analytes by detecting the color by visual observation or byusing a detecting device qualitatively or quantitatively, by using thefact that the hue (color) of the reaction line observed changesaccording to the abundance ratio of the substances A and B.

It is possible to analyze an analyte semi-quantitatively, by assumingnegative when the color tone is thinner than a particular color andpositive when it is thicker. Needless to say, the degree of colordevelopment may be determined, for example, by using a tester for urinetest paper such as Pretester RM-405, Pretester RM-505 (trade names,manufactured by Wako Pure Chemical Industries, Ltd.), a densitometer, orthe like.

Qualitative analysis is normally performed in advance to quantitativeanalysis, and the “semi-quantitative determination” herein above is aqualitative analysis slightly more quantitative numerically that isperformed before qualitative analysis.

Hereinafter, the immunochromatographic fluorescence-detecting system orthe radiation-detecting system of the present invention will bedescribed.

When silica nanoparticles containing a fluorescent substance as thelabelled substance are used as an immunochromatographic reagent, theimmunochromatographic fluorescence-detecting system of the presentinvention can be used similarly to the test strip described above.

In other words, the fluorescence-detecting system of the presentinvention has at least following components (1) and (2):

(1) a test strip comprising a sample pad, a member impregnated withfluorescent substance-containing silica nanoparticles (conjugate pad),an antibody-immobilized membrane and an absorption pad, and

(2) an excitation light source.

The test strip is preferably supported by a backing sheet.

In the fluorescence-detecting system of the present invention, theexcitation light source preferably emits an excitation light at awavelength of 200 nm to 400 nm, for detection of the fluorescence fromthe silica nanoparticles, for example, by visual observation. Examplesof the excitation light sources include mercury lamp, halogen lamp, andxenon lamp.

In addition, the fluorescence-detecting system of the present inventionmore preferably has a filter for transmission of a light at a particularwavelength from the excitation light source, and furthermore preferablyhas a filter transmitting only fluorescent light while blocking theexcitation light for detection only of fluorescence for example byvisual observation.

The fluorescence-detecting system of the present invention particularlypreferably has a photoelectron multiplier or CCD detector for detectionof the fluorescence, and it is possible in this way to detect afluorescence at an intensity or wavelength that is not detected byvisual observation. In addition, it is possible to performhigh-sensitivity detection and quantitative determination of an analyte,because the fluorescence intensity can be measured.

When silica nanoparticles containing a radioactive substance as thelabelled substance are used as an immunochromatographic reagent, theimmunochromatographic radiation-detecting system of the presentinvention can be used similarly to the test strip described above.

In other words, the radiation-detecting system of the present inventionhas at least following components (1) and (2):

(1) a test strip comprising a sample pad, a member impregnated withradioactive substance-containing silica nanoparticles (conjugate pad),an antibody-immobilized membrane and an absorption pad, and

(2) a Geiger counter or an X-ray film.

In the immunochromatographic radiation-detecting system of the presentinvention, it is possible to perform detection and quantitativedetermination of an analyte, by measuring radiation (radioactive ray)from the silica nanoparticles by means of a Geiger counter or byphotosensitizing radiation from the silica nanoparticles to an X-rayfilm

The labelled silica nanoparticles for immunochromatographic reagent ofthe present invention can provide various light absorptioncharacteristics, fluorescence characteristics or the like by varying alabelled substance contained therein, so that the labelled silicananoparticles for immunochromatographic reagent of the present inventioncan be used favorably in various immunochromatographic methods.

Further, the labelled silica nanoparticles for immunochromatographicreagent of the present invention do not offer blacken color, even if thelabelled silica nanoparticles aggregate or the like.

The immunochromatographic reagent of the present invention, whichemploys the labelled silica nanoparticles surface-modified with ananalyte-recognizing substance and thus provides various light absorptioncharacteristics, fluorescence characteristics or the like, can besuitably used in simultaneous analysis of multiple analytes.

The immunochromatographic test strip of the present invention, whichemploys the immunochromatographic reagent, can be used in simultaneousdetection and quantitative determination or judgment of multipleanalytes and thus, suitable as a new POCT diagnostic method.

Further, the immunochromatographic fluorescence-detecting system of thepresent invention, which employs silica nanoparticles containing afluorescent substance as the immunochromatographic reagent, allowshigh-sensitivity detection or quantitative determination by detection offluorescence.

Further, the immunochromatographic radiation-detecting system of thepresent invention, which employs silica nanoparticles containing aradioactive substance as the immunochromatographic reagent, allowshigh-sensitivity detection or quantitative determination by detection ofradiation.

EXAMPLES

The present invention will be described in more detail based on thefollowing examples, but the invention is not intended to be limitedthereto.

Reference Example 1 Preparation of Labelled Silica Nanoparticles for Usein the Present Invention

2.9 mg of 5- (and -6)-carboxytetramethylrhodamine succinimidyl ester(trade name, manufactured by emp Biotech GmbH) was dissolved in 1 ml ofdimethylformamide (DMF). 1.3 μl of APS was added thereto, and themixture was allowed to react at room temperature (23° C.) for 1 hour.

128 ml of ethanol, 400 μl of TEOS, 28.8 ml of distilled water, and 400μl of 28 mass % aqueous ammonia were added to 400 μl of the reactionsolution obtained, and the mixture was allowed to react at roomtemperature for 24 hours.

The reaction solution was centrifuged at a gravitational acceleration of18000×g for 30 minutes, and the supernatant was removed. Theprecipitated silica particles were redispersed in 4 ml of distilledwater, and the dispersion was centrifuged again at a gravitationalacceleration of 18000×g for 30 minutes. The above washing operation wasrepeated twice additionally, the unreacted TEOS and ammonia contained inthe labelled silica nanoparticles dispersion were removed, to give 100.8mg of silica nanoparticles having an average diameter of 104 nm (yield:approximately 94%).

FIG. 2 is a SEM micrograph showing the labelled silica nanoparticlesobtained. In the Figure, white spherical substances are the labelledsilica nanoparticles obtained.

Example 1 Detection of hCG

1 mL of 50 mM KH₂PO₄ (pH 7.5) and 8 mL of the rhodamine-containingsilica nanoparticle dispersion (6.25 mg/mL) were placed and agitatedgently in a centrifuge tube. 1 mL (5 mg/mL) of an anti-hCG antibody(Anti-hCG clone codes/5008, manufactured by Medix Biochemica) was addedinto the centrifuge tube while the mixture was agitated, and the mixturewas agitated at room temperature for 1 hour, allowing the anti-hCGantibody to be adsorbed on the silica nanoparticles. 100 μL of thereaction solution was placed in a microtube, and designated as colloidA. Residual of the reaction solution was designated as colloid B.

The colloid A was centrifuged at 12000×g for 15 minutes, and thesupernatant was collected. The concentration of the anti-hCG antibodycontained in the supernatant, as determined by the BCA method, was 239μg/ml. Because the concentration of the anti-hCG antibody at the startof reaction was 500 μg/ml and the weight concentration of the silicananoparticle was 5 mg/mL, 52.2 mg of anti-hCG antibody was bound to 1 gof the silica nanoparticles. The surface area of 1 g of the silicananoparticles, as calculated assuming that the density of the silicananoparticles is 2.3 g/cm³ and the particle diameter of the silicananoparticles 104 nm, is 25.1 m². Accordingly, the amount of theanti-hCG antibody bound to the silica nanoparticles per 1 m² of surfacearea was determined to be 2.1 mg.

0.55 mL of 1% PEG (polyethylene glycol, molecular weight: 20,000,manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto;the mixture was agitated gently; 1.1 mL of 10% BSA was addedadditionally; and the mixture was agitated gently.

The mixed liquid was centrifuged at 12000×g for 15 minutes; thesupernatant was removed as about 1 mL thereof was left behind; and thesediment was dispersed in the remaining supernatant. 20 mL of storagebuffer (20 mM Tris-HCl (pH 8.2), 0.05% PEG 20,000, 150 mM NaCl, 1% BSA,0.1% NaN₃) was added to the dispersion, and the mixture was centrifugedagain; the supernatant was removed, as about 1 mL of it was left behind;and the sediment was redispersed in the remaining supernatant. 1 mL ofdistilled water and 2 mL of a coating buffer (20 mM Tris-HCl (pH 8.2),0.05% PEG (molecular weight: 20,000), 150 mM NaCl, 1% BSA, 0.1% NaN₃)were added to the dispersion, and the mixture was agitated gently.

The dispersion of the silica nanoparticle carrying the adsorbed antibodythus obtained was applied uniformly on a sheet of Glass Fiber ConjugatePad (GFCP, manufactured by MILLIPORE) (8×150 mm) in an amount of 0.8 mL.The sheet was dried in desiccator at room temperature overnight underreduced pressure, to give a conjugate pad containing the silicananoparticles obtained in Reference Example 1.

An antibody-immobilized membrane was prepared by applying a solution((50 mM KH₂ PO₄, pH 7.0)+5% sucrose) containing 1 mg/mL of an anti-hCGantibody (α subunit of FSH (LH), clone code/6601, manufactured by MedixBiochemica) in a coating amount of 0.75 μL/cm, as a test line having awidth of approximately 1 mm in the central region of a membrane (Length:25 mm, product name: Hi-Flow Plus120 membrane, manufactured byMILLIPORE) (separated by approximately 12 mm from the edge).

Subsequently, a solution ((50 mMKH₂ PO₄, pH 7.0) sugar-free) containing1 mg/mL of an anti-IgG antibody (Anti Mouse IgG, manufactured by Dako)was coated thereon as a control line having a width of approximately 1mm in a coating amount of 0.75 μL/cm and the resulting membrane wasdried at 50° C. for 30 minutes.

Then, the membrane was immersed in a blocking buffer at room temperaturefor 30 minutes for blocking processing.

It was transferred into a membrane washing/stabilizing buffer and leftstill at room temperature for 30 minutes or more. The membrane waswithdrawn, placed on a paper towel, and dried at room temperatureovernight, to give an antibody-immobilized membrane.

The membrane obtained above, the conjugate pad obtained above, a samplepad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), andan absorption pad (Cellulose Fiber Sample Pad (CFSP), manufactured byMILLIPORE) were placed on a backing sheet (product name: AR9020,manufactured by Adhesives Research), and the composite was cut intostrips having a width of 5 mm and a length of 60 mm, to give test stripsin the configuration shown in FIGS. 1 a and 1 b. The FIGS. 1 a and 1 bare described above.

As shown in FIGS. 1 a and 1 b, respective constituent members wereplaced overlapped with the neighboring members at both ends to a widthof about 2 mm (hereinafter, the same shall apply).

When 50 IU/L of a recombinant hCG (manufactured by ROHTO PharmaceuticalCo., Ltd.) was applied dropwise onto the sample pad region of the stripin an amount of 100 μL and the strip was left for one minute, it wasconfirmed that the line containing the coated anti-hCG antibody (αsubunit of FSH (LH), clone code/6601, manufactured by Medix Biochemica)(test line) and the line containing the coated anti-IgG antibody(control line) developed red color.

Example 2 Detection of Multiple Biological Molecules with the Same Line

Rhodamine-containing silica nanoparticles were prepared in a similarmanner to Reference Example 1 (average diameter: 101 nm, yield: 90%).Separately, DYQ-660-containing silica nanoparticles were prepared in amanner similar to Reference Example 1 by using DYQ-660-NHS-Ester(manufactured by Dyomics GmbH). Subsequently in a similar manner toExample 1, the rhodamine-containing silica nanoparticles weresurface-modified with a mouse anti-influenza A virus nucleoproteinmonoclonal antibody by adsorption, and the DYQ-660-containing silicananoparticles were surface-modified with a mouse anti-influenza B virusnucleoprotein monoclonal antibody by adsorption.

0.8 mL of the dispersion containing the rhodamine-containing silicananoparticles surface-modified with mouse anti-influenza A virusnucleoprotein monoclonal antibody by adsorption was applied uniformly ona conjugate pad (Glass Fiber Conjugate Pad (GFCP), 8×150 mm,manufactured by MILLIPORE), and the pad was dried in desiccatorovernight under reduced pressure. Subsequently, 0.8 mL of a dispersioncontaining the DYQ-660-containing silica nanoparticles surface-modifiedwith mouse anti-influenza B virus nucleoprotein monoclonal antibody wasapplied uniformly on the same conjugate pad, and the pad was dried indesiccator overnight under reduced pressure. In this way, a conjugatepad impregnated both with rhodamine-containing silica nanoparticles andDYQ-660-containing silica nanoparticle was prepared.

Subsequently, an antibody-immobilized membrane was prepared by applyinga solution containing a mouse anti-influenza A virus nucleoproteinmonoclonal antibody and a mouse anti-influenza B virus nucleoproteinmonoclonal antibody respectively at 1 mg/mL, on the central region of amembrane (Length: 25 mm, product name: Hi-Flow Plus120 membrane,manufactured by MILLIPORE) (separated by approximately 12 mm from edge)as a test line having a width of approximately 1 mm in a coating amountof 0.75 μL/cm, and the resulting membrane was dried at 50° C. for 30minutes. Further, an anti-mouse antibody rabbit polyclonal antibody (1mg/mL) was coated in a coating amount of 0.75 μL/cm as the control line,and the membrane was dried at 50° C. for 30 minutes.

The membrane obtained above, the conjugate pad obtained above, a samplepad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), andan absorption pad (Cellulose Fiber Sample Pad (CFSP), manufactured byMILLIPORE) were placed on a backing sheet (product name: AR9020,manufactured by Adhesives Research); the composite was cut into stripshaving a width of 5 mm and a length of 60 mm, to give test strips in theconfiguration shown in FIGS. 1 a and 1 b. The FIGS. 1 a and 1 b aredescribed above.

Two of the test strips were made available, a solution containinginfluenza A virus at 5×10² FFU/mL was applied dropwise on one of them,and the strip was left still for one minute. A solution containinginfluenza B virus at 5×10² FFU/mL was applied dropwise on the other teststrip, and the strip was left still for one minute. The line on the teststrip where the influenza A virus-containing solution was applieddropwise turned red, while the sample line of the test strip where theinfluenza B virus-containing solution was applied dropwise turned blue.

Example 3 High-Sensitivity Detection of Fluorescence

Fluorescein-containing silica particles were prepared in a similarmanner to Reference Example 1 (average diameter: 93 nm, yield: 88%) andsurface-modified with an anti-hCG antibody (Anti-hCG clone codes/5008,manufactured by Medix Biochemica) by adsorption in a similar manner toExample 1. In a similar manner to Example 1, 0.8 ml of thefluorescein-containing silica nanoparticles surface-modified with theanti-hCG antibody by adsorption was coated on a Glass Fiber ConjugatePad (GFCP, 8×150 mm, manufactured by MILLIPORE), which was dried indesiccator overnight under reduced pressure. In this way, a conjugatepad containing fluorescein-containing silica nanoparticles was prepared.

In a similar manner to Example 1, a membrane (Hi-Flow Plus120 membrane,manufactured by MILLIPORE) carrying an anti-hCG antibody (α subunit ofFSH (LH), clone code/6601, manufactured by Medix Biochemica) as the testline and an anti-IgG antibody (Anti Mouse IgG, manufactured by Dako) asthe control line, a conjugate pad, an absorption pad (CFSP, manufacturedby MILLIPORE), and a sample pad (GFCP, manufactured by MILLIPORE) wereplaced on a backing sheet (product name: AR9020, manufactured byAdhesives Research); the composite was cut into strips having a width of5 mm and a length of 60 mm, to give test strips in the configurationshown in FIGS. 1 a and 1 b. The FIGS. 1 a and 1 b are described above.

100 μL of 0.5 IU/L recombinant hCG (manufactured by ROHTO PharmaceuticalCo., Ltd.) was applied dropwise on the sample pad of the test strip, andthe pad was left still for one minute.

The test strip was irradiated by a mercury lamp (103W), while a filterFF01-482 (trade name, manufactured by Semrock, Inc.) was used as thefilter in the excitation light source side and a filter FF01-536 (tradename, manufactured by Semrock, Inc.) as the filter in the detector side,and the imaging was performed by using a CCD detector (C2741-35 A (tradename, manufactured by Hamamatsu Photonics K.K.)) as the detector. As aresult, there was confirmed fluorescent emission from the test andcontrol lines.

The results show that the test strip in Example 3 allows detection ofanalyte (combinant hCG) in an amount of 1/100 of that observable byvisual observation in Example 1 indicating that it is possible toperform high-sensitivity detection with fluorescence.

Having described our invention as related to the present embodiments, itis our intention that the invention not be limited by any of the detailsof the description, unless otherwise specified, but rather be construedbroadly within its spirit and scope as set out in the accompanyingclaims.

What is claimed is:
 1. An immunochromatographic test strip, comprising asample application member, a member impregnated with animmunochromatographic reagent comprising labelled silica nanoparticleswhich are both surface-modified with an analyte-recognizing substanceand bonded with polyethylene glycol, a membrane having anantibody-immobilized region, and an absorption pad that are connected inseries.
 2. The immunochromatographic test strip according to claim 1,wherein two or more kinds of analytes are detected simultaneously, basedon the difference in hue of color formation or fluorescence wavelengthin a single antibody-immobilized region.
 3. An immunochromatographicfluorescence-detecting system, comprising: an immunochromatographic teststrip, comprising 1) a sample application member, 2) a memberimpregnated with an immunochromatographic reagent comprising labeledsilica nanoparticles, wherein said labeled silica nanoparticles are bothsurface-modified with an analyte-recognizing substance and bonded withpolyethylene glycol, 3) a membrane having an antibody-immobilizedregion, and 4) an absorption pad, wherein 1)-4) are connected in series,and wherein the immunochromatographic reagent allows high-sensitivitydetection by detection of fluorescence emitted from the silicananoparticles containing the fluorescent substance, and wherein thewavelength of an excitation light source used for detection of thefluorescence emitted from the silica nanoparticles is 200 nm to 600 nm.4. The immunochromatographic fluorescence-detecting system according toclaim 3, further comprising a filter eliminating the excitation lightand transmitting only the fluorescence.
 5. The immunochromatographicfluorescence-detecting system according to claim 4, further comprising aphotoelectron multiplier or a CCD detector detecting the fluorescence,wherein the system allows quantitative determination of the analyte bydetection of the fluorescence and measurement of the fluorescentintensity with the photoelectron multiplier or the CCD detector.
 6. Theimmunochromatographic fluorescence-detecting system according to claim3, wherein the excitation light source is a mercury lamp, a halogenlamp, or a xenon lamp.
 7. An immunochromatographic radiation-detectingsystem, comprising: an immunochromatographic test strip, comprising 1) asample application member, 2) a member impregnated with animmunochromatographic reagent comprising labelled silica nanoparticles,wherein said labelled silica nanoparticles are both surface-modifiedwith an analyte-recognizing substance and bonded with polyethyleneglycol, 3) a membrane having an antibody-immobilized region, and 4) anabsorption pad, wherein 1)-4) are connected in series, and whereinradiation emitted from the silica nanoparticles is measured with aGeiger counter or by photosensitizing an X-ray film.